NCBI Gene # or RefSeq#: folK FTT_0942c (gene name) 3191528 (gene ID)
Protein ID (NP or XP #) or Wolbachia#: Mmdb_id: 121548
Organism (including strain): Francisella tularensis (F. tularensis tularensis)
Etiologic Risk Group (see link below): Category A NIAID Biodefense Research
Disease Information (sort of like the Intro to your Mini Research Write up):
Tularemia (AKA Pahvant Valley plague, rabbit fever, deer fly fever, and Ohara's fever) is caused by Francisella Tularensis, a gram negative bacteria. Disease found in Rabits, Hairs, Pikas, small rodents, and Humans. Spread by ticks, deer flies, some arthropods, and ingestion of contaminated water or food.
The incubation period for the disease is 14 days, clinical symptoms in humans appear after 3 and include: fever, lethargy, loss of appetite, signs of sepsis, enlargement of the lymphnodes (similar to the bubonic plague) and possibly death. Overall fatality for untreated cases is 7%, however some strains are more virulent.
Essentiality of this protein:
Statement of essentiality: “The marginal DHPS activity and the single copy existence of FtHPPK-DHPS in F. tularensis make this bacterium more vulnerable to DHPS inhibitors. Current sulfa drugs are ineffective against tularemia; new inhibitors targeting the unique pABA-binding pocket may be effective and less subject to resistance because any mutations introducing resistance may make the marginal DHPS activity unable to support the growth of F. tularensis.” From: http://dx.doi.org/10.1111/febs.12896
Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism): Examined in E. Coli, so druggable in E. Coli. From article found at link: http://onlinelibrary.wiley.com/doi/10.1111/febs.12896/full
-- links to assay reagents (substrates) pages
Final concentrations of reagents in the standard assay were as follows: 50 mM Tris–HCl (pH 8.5), 1.0 mg/ml bovine serum albumin, 5.0 mM MgCl2, 20 mM β-mercaptoethanol, 50 mM NADPH, 50 mM 6-hydroxymethyl-7,8-dihydropterin or the pyrophosphate, 50 mM p-ABA, and 1.0 μg/ml DHFR. ATP (final concentration 100 μM).
Substrate: 4-aminobenzoate
From: http://www.sciencedirect.com/science/article/pii/S0003269706007809
--- List cost and quantity of substrate reagents, supplier, and catalog #
Couldn’t fine 4 aminobenzoate for sale, however Ethlyl4-aminobenzoate is marketable, esterhydrolisis could be done to remove the Ethyl.
Cost: $29.20
Quantity: 5 grams
Suplier: Sigma-Aldrich
Catalog #: 112909
Structure (PDB or Homology model) -- PDB # or closest PDB entry if using homology model: 4PVZ
Figure 2: PDB image of enzyme HPPK/DHPS, homology model: 4PVZ
Current Inhibitors:
HPPK/DHPS in plants feedback inhibited by dihydropteroate
Methotrexate inhibits just DHPS
Possibility of pterin based inhibitors
Article looking into new class of inhibitors that targets the pABA binding sight to inhibit both HPPK/DHPS: http://www.sciencedirect.com/science/article/pii/S0003269706007809
Expression Information (has it been expressed in bacterial cells): Yes, in E.Coli
Purification Method: metal affinity chromatography and ion exchange chromatography Image of protein (PyMol with features delineated and shown separately):
Figure 3: Pymol representation of HPPK/DHPS showing surface. color coded as carbon green Figure 4: Pymol Representation of HPPK/DHPS as cartoon. Carbon green.
Oxygen red, and nitrogen blue.
Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
MQYIIGIGTNIGFTIENIHLAITALESQQNIRIIRKASLYSSKAVLKEDAPKEWDIRFLNTAVKISSSLKPDELLVLLKD
IELKIGRDLNAPAWSPRVIDLDILAAEDLILETDKLTIPHKELINRSFALAPLLELSKGWHHPKYVEWDLNIRLKELGEI
VKLKQTLANTIRMGIVNLSNQSFSDGNFDDNQRKLNLDELIQSGAEIIDIGAESTKPDAKPISIEEEFNKLDEFLEYFKS
QLANLIYKPLVSIDTRKLEVMQKILAKHHDIIWMINDVECNNIEQKAQLIAKYNKKYVIIHNLGITDRNQYLDKENAIDN
VCDYIEQKKQILLKHGIAQQNIYFDIGFGFGKKSDTARYLLENIIEIKRRLELKALVGHSRKPSVLGLAKDSNLATLDRA
TRELSRKLEKLDIDIIRVHKI
length of your protein in Amino Acids: 421
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website:
48243.9 Da
Molar Extinction coefficient of your protein at 280 nm wavelength:
44015
CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
GC% Content for gene (codon optimized):
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to. Primer design results for 'tail' primers (this is just 2 sequences):
Target (protein/gene name): HPPK-DHPS (2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase)
NCBI Gene # or RefSeq#: folK FTT_0942c (gene name) 3191528 (gene ID)
Protein ID (NP or XP #) or Wolbachia#: Mmdb_id: 121548
Organism (including strain): Francisella tularensis (F. tularensis tularensis)
Etiologic Risk Group (see link below): Category A NIAID Biodefense Research
Disease Information (sort of like the Intro to your Mini Research Write up):
Tularemia (AKA Pahvant Valley plague, rabbit fever, deer fly fever, and Ohara's fever) is caused by Francisella Tularensis, a gram negative bacteria. Disease found in Rabits, Hairs, Pikas, small rodents, and Humans. Spread by ticks, deer flies, some arthropods, and ingestion of contaminated water or food.
The incubation period for the disease is 14 days, clinical symptoms in humans appear after 3 and include: fever, lethargy, loss of appetite, signs of sepsis, enlargement of the lymphnodes (similar to the bubonic plague) and possibly death. Overall fatality for untreated cases is 7%, however some strains are more virulent.
Link to TDR Targets page (if present): NA
Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.)
http://www.ncbi.nlm.nih.gov/protein/4PZV_A
Essentiality of this protein:
Statement of essentiality: “The marginal DHPS activity and the single copy existence of FtHPPK-DHPS in F. tularensis make this bacterium more vulnerable to DHPS inhibitors. Current sulfa drugs are ineffective against tularemia; new inhibitors targeting the unique pABA-binding pocket may be effective and less subject to resistance because any mutations introducing resistance may make the marginal DHPS activity unable to support the growth of F. tularensis.” From: http://dx.doi.org/10.1111/febs.12896
Is it a monomer or multimer as biological unit? (make prediction at http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html): Monomer
Complex of proteins?: NA
Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism): Examined in E. Coli, so druggable in E. Coli. From article found at link: http://onlinelibrary.wiley.com/doi/10.1111/febs.12896/full
EC#: 2.5.1.15
Link to BRENDA EC# page: http://www.brenda-enzymes.org/enzyme.php?ecno=2.5.1.15
Figure 1: Reaction schematic catalyzed by enzyme HPPK/DHPS
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents): coupled, enzymatic spectrophotometric assay
-- -or link (or citation) to paper that contains assay information http://www.sciencedirect.com/science/article/pii/S0003269706007809
-- links to assay reagents (substrates) pages
Final concentrations of reagents in the standard assay were as follows: 50 mM Tris–HCl (pH 8.5), 1.0 mg/ml bovine serum albumin, 5.0 mM MgCl2, 20 mM β-mercaptoethanol, 50 mM NADPH, 50 mM 6-hydroxymethyl-7,8-dihydropterin or the pyrophosphate, 50 mM p-ABA, and 1.0 μg/ml DHFR. ATP (final concentration 100 μM).
Substrate: 4-aminobenzoate
From: http://www.sciencedirect.com/science/article/pii/S0003269706007809
--- List cost and quantity of substrate reagents, supplier, and catalog #
Couldn’t fine 4 aminobenzoate for sale, however Ethlyl4-aminobenzoate is marketable, esterhydrolisis could be done to remove the Ethyl.
Cost: $29.20
Quantity: 5 grams
Suplier: Sigma-Aldrich
Catalog #: 112909
Structure (PDB or Homology model)
-- PDB # or closest PDB entry if using homology model: 4PVZ
Figure 2: PDB image of enzyme HPPK/DHPS, homology model: 4PVZ
Current Inhibitors:
HPPK/DHPS in plants feedback inhibited by dihydropteroate
Methotrexate inhibits just DHPS
Possibility of pterin based inhibitors
Article looking into new class of inhibitors that targets the pABA binding sight to inhibit both HPPK/DHPS: http://www.sciencedirect.com/science/article/pii/S0003269706007809
Expression Information (has it been expressed in bacterial cells): Yes, in E.Coli
Purification Method: metal affinity chromatography and ion exchange chromatography
Image of protein (PyMol with features delineated and shown separately):
Figure 3: Pymol representation of HPPK/DHPS showing surface. color coded as carbon green Figure 4: Pymol Representation of HPPK/DHPS as cartoon. Carbon green.
Oxygen red, and nitrogen blue.
Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
MQYIIGIGTNIGFTIENIHLAITALESQQNIRIIRKASLYSSKAVLKEDAPKEWDIRFLNTAVKISSSLKPDELLVLLKD
IELKIGRDLNAPAWSPRVIDLDILAAEDLILETDKLTIPHKELINRSFALAPLLELSKGWHHPKYVEWDLNIRLKELGEI
VKLKQTLANTIRMGIVNLSNQSFSDGNFDDNQRKLNLDELIQSGAEIIDIGAESTKPDAKPISIEEEFNKLDEFLEYFKS
QLANLIYKPLVSIDTRKLEVMQKILAKHHDIIWMINDVECNNIEQKAQLIAKYNKKYVIIHNLGITDRNQYLDKENAIDN
VCDYIEQKKQILLKHGIAQQNIYFDIGFGFGKKSDTARYLLENIIEIKRRLELKALVGHSRKPSVLGLAKDSNLATLDRA
TRELSRKLEKLDIDIIRVHKI
length of your protein in Amino Acids: 421
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website:
48243.9 Da
Molar Extinction coefficient of your protein at 280 nm wavelength:
44015
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html):
Figure 5: TMPred graph of HPPK/DHPS enzyme.
CDS Gene Sequence (paste as text only):
GTGCAATATATTATAGGAATTGGAACAAATATTGGTTTTACTATCGAAAATATTCATCTTGCAATAACTG
CACTAGAATCGCAACAAAATATAAGAATCATCAGAAAAGCAAGCTTATACAGTAGTAAAGCTGTCCTTAA
AGAAGACGCTCCTAAAGAATGGGATATTAGATTTTTAAATACAGCTGTAAAAATTAGCTCTTCACTAAAG
CCTGATGAACTTTTAGTACTCTTAAAAGACATAGAATTAAAAATAGGTAGAGACCTAAATGCTCCTGCAT
GGTCACCTCGAGTAATTGATTTAGATATTCTTGCTGCTGAAGATCTAATTTTAGAAACAGACAAACTTAC
TATTCCTCATAAAGAATTAATTAATCGTAGTTTTGCGTTGGCTCCTTTATTAGAATTATCAAAGGGTTGG
CATCACCCTAAATATGTTGAATGGGATCTTAATATAAGATTAAAAGAATTAGGTGAGATAGTAAAACTTA
AACAAACTCTTGCAAATACAATACGTATGGGTATAGTTAATCTGTCAAATCAATCTTTTTCAGATGGTAA
TTTTGATGATAATCAACGTAAATTAAACCTTGATGAGCTAATACAAAGTGGTGCTGAAATTATTGATATC
GGAGCTGAATCGACTAAGCCTGATGCCAAGCCTATATCGATTGAAGAAGAATTTAACAAATTAGATGAAT
TCCTAGAATACTTTAAATCACAACTGGCAAATTTGATTTATAAACCATTAGTCAGTATTGACACACGCAA
ACTAGAGGTAATGCAAAAAATTCTCGCTAAGCATCATGATATTATCTGGATGATAAATGATGTTGAATGT
AATAATATTGAGCAAAAAGCACAGCTTATAGCTAAATATAATAAAAAGTATGTTATAATTCATAATTTGG
GTATTACAGATAGAAATCAATATTTAGATAAAGAAAATGCTATAGATAATGTTTGTGATTATATTGAGCA
AAAAAAGCAAATTCTTCTAAAACATGGTATAGCACAACAAAATATTTATTTTGATATTGGCTTTGGTTTT
GGTAAAAAATCAGATACCGCTAGATACTTATTAGAGAATATCATCGAGATAAAAAGAAGATTAGAATTAA
AAGCATTAGTTGGTCACTCACGTAAGCCATCAGTTTTAGGATTAGCAAAAGATAGTAATTTAGCAACTTT
AGATCGAGCTACAAGAGAGCTTTCAAGAAAATTAGAGAAACTAGATATTGATATTATAAGAGTACACAAG
ATTTAA
GC% Content for gene:
29.69%
CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
GC% Content for gene (codon optimized):
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):